Non-alcoholic liver steatosis, also called fatty liver, is due to excessive fat accumulation in hepatocytes and is the most common liver disease affecting 10-30% of the general population. Fatty liver is also regarded as a component of metabolic syndrome, and is associated with insulin resistance, obesity, and diabetes. Nearly 35% of patients with fatty liver develop chronic inflammation, fibrosis, and eventual liver failure and there is no effective treatment for fatty liver disease. Therefore, it is imperative to understand the molecular mechanisms underlying the development of fatty liver. Our preliminary studies demonstrate that the damage-specific DNA binding protein 1 (DDB1) E3 ligase plays a crucial role in the development of high-fat diet (HFD)-induced fatty liver. In particular, we discovered that liver DDB1 protein levels were elevated under obese conditions. Liver-specific deletion of Ddb1 protects mice from the HFD-induced fat accumulation and selectively suppresses glycolytic and lipogenic gene expression in the liver. In primary mouse hepatocytes, Ddb1 knockdown represses de novo lipogenesis rate, reduces triglyceride content, and blocks the activity of a key regulator of lipogenesis, carbohydrate responsive- element binding protein (ChREBP). At the mechanistic level, we uncovered evidence that DDB1 indirectly regulates the ChREBP protein O-GlcNAcylation and stability by degrading O-GlcNAcase (OGA) through ubiquitination. Although DDB1 has been well-studied in the context of DNA damage response as a critical component of the CUL4A E3 ligase complex, its metabolic actions remain unknown. Based upon the preliminary data, we hypothesize that DDB1 E3 ligase contributes to the development of fatty liver by activating ChREBP-mediated hepatic lipogenesis. To test this hypothesis, we propose three inter- connected specific aims. Aim 1: Determine whether the DDB1 E3 ligase promotes de novo lipogenesis and contributes to obesity-associated fatty liver via ChREBP. Aim 2: Test whether DDB1 E3 ligase promotes O- GlcNAcylation and stability of ChREBP via degrading OGA protein in obesity. Aim 3: Investigate how the AMPK pathway mediates nutritional regulation of hepatic DDB1 expression during the obesity development. This proposal seeks an in-depth understanding of the metabolic role of DDB1 E3 ligase. Specifically, we anticipate identifying how DDB1 E3 ligase regulates de novo lipogenesis in the liver and induces fatty liver through ChREBP. Accomplishment of these research objectives should reveal fundamental mechanisms by which nutrient excess leads to fat accumulation and provide potential novel therapeutic avenues to treat fatty liver disease.